Chapter 3 72 Framework Criteria What to report Please complete each criterion 4.1 Trigger Channel(s) and Threshold(s). The trigger channel(s) and threshold(s) used for event detection. Preferably, the fluorescence calibration (Section 4.3) and/or scatter calibration (Section 4.4) should be used in order to report the trigger channel(s) and threshold(s) in standardized units. Based on unstained, single-stained and isotype control samples, detection for CFSE fluorescence was triggered with 488nm laser at full power (200 mW), detected in channel 2 (480-560 filter) at a threshold of 170 arbitrary units, equivalent to ~36 FITC ERF, deterimined using Spherotec Rainbow Calibration beads and the manufacturers calibration values. Similarly, APC fluorescence was triggered with 642 nm laser at full power (150 mW), detected in channel 5 (642-745 filter), at a threshold of 170 a.u., equivalent to ~6 ERF APC. BV421 fluorescence was triggered with 405 nm laser at full pwer (120 mW), detected in channel 1 (435-505 nm filter), at a threshold of 110 a.u., equivalent to ~678 C30 ERF. 4.2 Flow Rate / Volumetric quantification. State if the flow rate was quantified/validated and if so, report the result and how they were obtained. Flow speed was monitored during acquisition and acquisition was started when flow speed was between 43.5 - 43.7 mm/sec. Typically, with the IFCM set at ‘low speed, high sensitivity’, ~0.8 uL of sample was measured in the time span of 180 seconds. 4.3 Fluorescence Calibration. State whether fluorescence calibration was implemented, and if so, report the materials and methods used, catalogue numbers, lot numbers, and supplied reference units for the standards. Fluorescence parameters may be reported in standardized units of MESF, ERF, or ABC beads. The type of regression used, and the resulting scatter plot of arbitrary data vs standard data for the reference particles should be supplied. Arbitrary BV421, CFSE and APC fluorescence intensities were converted to ERF units using 500 nm Rainbow Calibration Particles (RCP-05-5, lot AL01, Spherotech) with known Equivalent number of Reference Fluorophores (ERF) values for C30/FITC/APC. For each detection channel, the MFI of each fluorescent peak (blanc peaks were omitted from the regression analysis) from the four bead populations (1 blanc – 3 fluorescent) were measured, and a linear regression analysis was performed of the log(10) of these values against the log(10) of the known ERF values. The resulting equations were used to convert BV421/CFSE/APC fluorescent intensities into ERF units.
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